PROJECT SUMMARY The mechanisms that regulate the species composition and functions of vaginal microbiota in health and disease are not well understood and constitute a fundamental knowledge gap. Dysbiosis of vaginal microbiota leads to the symptoms associated with bacterial vaginosis (BV); a disease common in reproductive-age women that results in millions of health care visit annually and increases risks to the acquisition of sexually transmitted infections and adverse pregnancy outcomes. Unfortunately, interventions for the treatment of BV are often not effective and recurrence is common. After decades of looking and not identifying one particular bacterial taxa as the causative factor of BV, researchers have come to the consensus that BV is essentially an ecological disease in which shifts in the composition and structure of vaginal bacterial communities are linked with disease symptoms. However, the underlying biological trigger, which sets vaginal bacterial communities on the path to a BV state, is unknown. Thus, the central hypothesis of the proposed research is that bacteriophage infection is a contributing underlying biological causative factor capable of rapidly altering the composition of vaginal bacterial communities, changes that ultimately lead to BV. Numerous investigations of natural environments, and to a more limited extent investigations of the microbial environments of the human body, tell us that communities of free DNA viruses contain a diverse collection of extraordinarily abundant viral populations. These viral communities are composed predominantly of bacteriophages, i.e., bacterial viruses. As efficient and specialized bacterial predators, phages have the potential to rapidly decimate bacterial populations and thus alter the overall composition of co-occurring bacterial communities. To our knowledge no investigations have utilized cultivation-independent tools for direct examination of phage-host dynamics within the vagina. We will test this hypothesis by pursuing two specific aims: 1) Characterize fluctuations in the activity of CRISPR/Cas systems and the diversity of CRISPR spacer elements prior to, the day of disease onset, and during BV events; and 2) determine whether changes in the abundance and composition of free vaginal bacteriophage communities are linked to the onset of BV. To achieve these aims we will leverage a unique set of samples and metadata that were self-collected daily by 135 women for 10 weeks. During this period, 20 women experienced symptomatic BV that required medical treatment. This affords us a unique opportunity to examine the ecological interactions of vaginal phage and bacterial communities across BV events using state-of-the-art genomic technologies. The rationale for the proposed research is that exploring and understanding the biological mechanisms leading to BV has the potential to translate to effective means to insure vaginal homeostasis. The long-term goal of the proposed research is to contribute to a better understanding of the causes of BV, the homeostatic mechanisms at work in the human vagina, and to devise strategies to minimize dysbiosis and the risks associated with BV. !